Method for the transformation of cells, particularly eukaryotes by a DNA originating from viruses of hepatitis, more particularly from virus of B viral hepatitis, and preparations containing the expression products of said DNAs

ABSTRACT

Method for the production of antigens vaccinating against the virus of B viral hepatitis. It consists of transforming a cell culture with a vector, more particularly a plasmid, itself containing an insertion sequence including itself at least the part of the viral DNA coding for the immunogen protein, capable of inducing in vivo antibody production active with respect to the whole virus, as well as the viral promoter under the control of which the transcription and translation of the above-said part of viral DNA is normally carried out, in particular in a host infected with the corresponding virus.

This application is a continuation of application Ser. No. 07/900,483,filed Jun. 18, 1992, now abandoned which is a division of applicationSer. No. 06/482,322, filed Apr. 5, 1983, now U.S. Pat. No. 5,314,808which is a continuation of application Ser. No. 06/256,126, filed Apr.21, 1981 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a preparation containing antigens possessingimmunological properties, notably antigenic properties, characteristicof viruses of various forms of hepatitis, such as B viral hepatitis orvarious other forms of hepatitis such a those which are known to bedeveloped in certain patients following blood transfusions, for examplehepatitides called "non A" or "non B". The invention relates moreparticularly to preparations of this type, which are characterised byhigh purity, the absence of proteins of human origin and, when itconcerns preparations having antigen properties similar to those of theHBs antigens, free of Dane particles. It also relates to a method forthe production of these antigens.

2. Description of the Prior Art

It is known that certain of the specific antigen properties of the virusof hepatitis must be attributed to an antigen called "HBs Antigen" or"HBsAg", essentially formed by the envelope of the virus of viralhepatitis of the B type (HBV) or Dane particle. This antigen, whichpossesses vaccinating properties with respect to type B viral hepatitis,is, at the present time, essentially obtained from human serumspecimens. However, no other sources for the supply of HBsAg, are by nowavailable by reason of the particular characteristics of the virus of Bhepatitis (HBV). It seems only capable of infecting man, the chimpanzeeand, perhaps, a small number of other primates. It has not been possiblehitherto to propagate it in vitro in cell cultures. Certain lines ofhepatocarcinomas which synthesize HBsAg are known. The use in man ofcancerous cells for the production of particles with a vaccinatingcharacter runs up against quite comprehensible objections. The use inpreventive therapy of HBs antigens of human origin is not however devoidof serious risks. In fact, they arise generally from persons who havebeen exposed to the virus of B viral hepatitis, so that the presence ofDane particles, sometimes also highly infectious, in preparations ofHBsAg antigens of serum origin cannot always be completely excluded,even in the case of extremely pure preparations. The contents of theeven highly purified preparations of HBsAg antigen from the state of theart in serum proteins or other possibly antigenic components, capable ofinducing in the treated subjects troublesome immunological reactions,are not negligible, by reason of their very low initial content of HBsAgwith respect to the proteins of the initial serum (for example, of theorder of 50 μg of HBsAg with respect to 80 mg of protein in a 1 ml ofserum).

In spite of the difficulties encountered to have available sufficientamounts of virus, it has however been established that the genome of thevirus of B hepatitis is formed from a partially single stranded circularDNA molecule, of which the longest strand includes of the order of 3200nucleotides (SUMMERS J. and coll. (1975) Proc. Nat. Sci. U.S.A. 72,4597-4601). At the most, it has been possible to localise the genecoding the portion of the protein of the HBsAg antigen, which isresponsible for the immunological properties of the envelope of thevirus of B hepatitis. The position of this gene, named "S gene" resultsnotably from the diagrammatic map of the genome of the Dane particle,which is given in FIG. 1 of the drawings.

FIG. 1 comprises a diagrammatic map of the DNA of the genome of the Daneparticle. This DNA comprises two strands b₁ and b₂, the shortest of them(b₂) being normally devoid of the portion shown by a dashed line in thedrawing.

It is known that this DNA only includes a single EcoRI site.

The arrow f₁ gives the direction of the numbering of the nucleotidesfrom which the longest strand b₁ is composed, and the arrow f₂ gives thedirection of the transcription of the S gene by the cellular machineryof the cells invaded by the virus of B hepatitis.

The EcoRI site can hence be numbered 0, or 3182 (in the case of Bhepatitis viruses belonging to serotype 3182) (NATURE, 1979, vol. 281,p. 646-650).

The inner concentric circle e₁ gives the scale in numbers ofnucleotides. This circle enables the positions of certain of the partsof this DNA to be specified. The numbers 3', 5' and 5', 3', at the lowerpart of the map indicate the terminal ends bearing the same numbers inconventional representations of the ends of the nucleic acid chains.

At the most, the coding gene for the part of the protein of the HBsAgantigen has been localised. The position of this gene, named "S gene",is shown diagrammatically by the arrow S in the Figure. The "S gene" isessentially borne by the fragment of the longest strand b₁ situatedbetween the nucleotide positions 155 and 833 of the diagrammatic map ofFIG. 1 in the direction of the transcription of the S gene.

The shortest strand (b₂) of the genome of the Dane particle can be"repaired" in vitro in the presence of precursor nucleotides and of apolymerase, for example by the technique of T. A. LANDERS and coll., J.VIROL., 23, 1977, p. 368-376. The genomes so-repaired of the viralhepatitis viruses (or any DNA capable of coding for the same aminoacidsequences) will be denoted below by the abbreviation DNA HBV.

It is therefore an object of the invention to provide processes such ashave been specified above, which are applicable generally to the studyand to the expression in eukaryotic cells of all or part of the wholegenomes of the viruses responsible for the various viral hepatites, moreparticularly of DNA HBV. It is also an object to provide for theproduction of modified vectors enabling the practising of these methods.

Lastly, it is more particularly an object of the invention to providefor the production of preparations containing antigens having the sameimmunological specificity as HBsAg or of a similar antigen of highpurity, as has been indicated above, from a system other than humanserum, which system is both reproducible and stable (or having a certainnumber of particular characteristics which can be made the subject ofconstant surveillance).

SUMMARY OF THE INVENTION

According to the invention there is provided a method for determiningthe eventual possibility of transformation of a culture of particularcells by a DNA normally circular or a whole genome of the correspondingvirus, which method comprises the operation which consists oftransforming this cell culture with a vector, more particularly aplasmid, itself containing an insertion sequence including itself atleast the portion of the viral DNA coding for the immunogenic protein,capable of inducing in vivo the production of active antibodies withrespect to the whole virus, as well as the viral promoter under thecontrol of which the transcription and the translation of the above-saidportion of viral DNA is normally carried out, in particular in a hostinfected by the corresponding virus.

According to a first embodiment of the invention, the above-saidinsertion sequence is formed by at least two of these DNAs, such as twoDNA HBVs, oriented in the same direction of transcription and in whichthe tail of one is connected to the head of the other. In the following,there will often be used, for convenience of language, the expression"tandem sequence", to denote insertion sequences comprising two DNAs ofthe type concerned, except when otherwise specified.

In a preferred embodiment of the method according to the invention, theabove-said insertion sequence is formed from at least two of these DNAHBVs or from two fragments of DNA HBV, normally contained in DNA HBV oneach side of its EcoRI site, oriented in the same direction oftranscription, one of these fragments of DNA HBV containing the sequenceof nucleotides of the S gene, which is adapted to code for thepolypeptide responsible for the immunological properties of the HBsantigen and the other of said fragments being sufficiently long for itto be capable of including the promoter of the S gene.

According to a first preferred embodiment of the process according tothe invention, the oriented DNAs of the insertion sequence areconstituted by fragments of DNA HBV, the tail of one of these fragmentsbeing connected to the head of the other fragment at the level of anEcoRI site.

It is self-evident that certain non essential parts of the latterinsertion sequences can be deleted. Such an insertion sequence containstherefore advantageously, on the one hand, the DNA HBV fragment whichcontains the nucleotide sequence of the S gene, coding for thepolypeptide responsible for the immunological properties of the HBsantigen and, on the other hand, a DNA HBV fragment being sufficientlylong for it to be capable of including the promoter of the S gene,upstream of the preceding fragment in the direction of transcriptionthese two fragments being normally situated on each side of the DNA HBVEcoRI site.

In other words, this insertion sequence comprises notably, if one refersto FIG. 1, on the one hand, the sub-fragment comprised between thenucleotide positions 833 to 0 (direction reverse to f₁), when thissub-fragment comprises the S gene as a whole and, on the other hand, asub-fragment comprised between the position 3182 and a positioncorresponding to a number sufficiently distant from this 0 position, inthe reverse direction of the transcription, so that the above-saidpromoter can be included therein.

It is observed that the last sub-fragment must normally comprise aTATATAA sequence, which is normally found upstream of the beginning ofthe S gene, at a distance corresponding to a number of nucleotides ofthe order of 235. More particularly again, the insertion sequence willcomprise an additional portion of the DNA HBV situated upstream of theBglII site, itself at the level of the nucleotide 2840.

According to an additional feature of the invention, the insertionsequence comprises also in addition a sub-fragment downstream of the Sgene in the direction of transcription, this fragment extending normallyin the direction of the transcription beyond the DNA HBV BamHI site.

A preferred insertion sequence comprises the whole of the coding genefor the messenger RNA corresponding to the HBs antigen. The relativeposition of this RNA messenger is represented in FIG. 1 by the arrow ARNof which the beginning coincides substantially with the position of thenucleotide 2800, of one refers to the map of FIG. 1, and whichterminates between the BamH₁ and BGlII 1986 site.

A preferred insertion sequence capable of being applied in the methodaccording to the invention is hence characterised in that it comprisesboth the S gene and the coding gene for the messenger RNA correspondingto the HBs antigen. As is to be concluded from the rest of thisdescription, this sequence is preferably devoid of any part of thecoding gene for the HBc antigen, sufficing to be expressed in the formof a protein having the immunogenic properties of the HBc antigen.

In the rest of this description, reference will be made moreparticularly to DNA HBV. It is nonetheless understood that all thetechniques which are described are also applicable to the DNA orcorresponding whole genomes of other viruses, responsible for othertypes of hepatitis.

Preferably, conditions are adopted which permit at the same timelabelling of the transformed cells, so as to render their detectioneasy, for example, by using cells or mutants of these cells rendereddeficient, for selection purposes (natural or induced deficiency), of aselectable labelling gene normally necessary for their growth, when theyare placed in certain particular culture media, these cells beingnevertheless capable of developing again in one at least of these sameculture media, after introduction in these cells of a gene or fragmentof homologous DNA, although of foreign origin (complementation DNA),capable of compensating or "complementing" said deficiency, the methodconsisting then, either of attempting to carry out the transformation ofsaid cells:

either with a single vector containing, on the one hand, the insertionsequence studied, notably derived from DNA HBV, and on the other hand,such a "complementation DNA", respectively inserted in its own genome,

or with two vectors (simultaneous transformations, orco-transformation),

one of the vectors, preferably a plasmid, having previously beenmodified by the insertion in its genome of such a complementation DNA;

the other vector, preferably also a plasmid, having previously beenmodified by the insertion in its genome of the abovesaid insertionsequence,

and of collecting, after their cultivation in the above-defined medium,of the colonies which both developed and which were transformed by theinsertion sequence.

To the extent that the DNA HBV is capable of being expressed in the cellof said culture, the tandem sequence (or containing more than two DNAHBV units), or more generally the insertion sequence such as has beendefined above, must normally behave as would the corresponding circularDNA thus introduced into the cell, in the absence of any vector.

The use of such a vector, notably a plasmid, instead and in place of thecircular DNA itself is of very great interest in that it is possible tohave considerable amounts of such a vector after amplification of thevector previously constructed in vitro by cloning in bacteria.

The process according to the invention hence permits testing thecapacity of a circular DNA or of a whole virus genome--in particularDNAs of hepatitis viruses other than DNA HBV--to be expressed ineukaryotic cells, to be tested, without it being necessary to giveregard to the exact position of the promoter under the control of whichthe transcription is to be effected.

The vectors modified by the above-said insertion sequences, notablythose derived from DNA HBV can be used to induce the expression of theseinsertion sequences in eukaryotic cell cultures, notably of the mouse orof human origin.

When there is used, for the labelling of transformed cells, a distinctvector, notably a plasmid containing the marker, it is preferable toresort to a plasmid containing a "complementation DNA", such as the geneof thymidine-kinase of the Herpes simplex HSV-1 virus, which can beexcised from the genome of the virus by specific enzymes restrictionssuch as BamHI. To this gene of thymidine-kinase of viral origincorrespond, in numerous types of eukaryotic cells, homologous genesadapted to direct the synthesis of the thymidine-kinase (enzymephosphorylating the exogenous thymidine supplied by the culture medium).

The possiblity of overcoming certain genetic deficiencies of eukarioticcells, notably of the mouse, for example, those relating to their geneof endogenous thymidine-kinase, by reason of an induced or provokedmutation, has already been described by M. WIGLER and coll. (Cell vol.II, 223-232, 1977). The deficient cells, called TK⁻, are selected byreason of their incapacity to synthesize thymidine kinase in a mediumsuch as that known by the name "HAT medium"(containing hypoxanthine andaminopterine in addition to thymidine). This medium is known for onlypermitting the possible synthesis of thymidine phosphate via a metabolicroute called "salvation pathway", this route implies however that theintegrity of the TK gene of the cells capable of being developed thereinbe preserved. TK⁻ cells become nonetheless capable of development inthis same medium as soon as they have been modified by incorporation ofthe TK gene of the virus of the herpes which is then geneticallytransmissible to their descendants, as a result of successive celldivisions. From "TK⁻ " as they were previously, before the abovesaidincorporation, these cells have then become "TK⁺ ", due to the fact ofthe restoration then observed of their capacity to phosphorylatethymidine in the abovesaid medium and consequently of developingtherein.

Of course, the DNA fragment containing the gene of thymidine kinase canbe replaced by any other suitable complementation DNA. By way of exampleof other complementation genes which can be used for the constitution ofthe vectors according to the invention, will be mentioned thatcontaining that of dihydrofolate reductase (DHFR), or in general, anycomplementation gene of which numerous examples exist in nature.

The complementation DNA may naturally include a natural gene. It mayalso be synthesized, notably enzymatically, by copying a correspondingmessenger RNA.

As regards the technique of co-transformation itself, it is advantageousto use the vector in which the complementation gene is inserted and thevector in which the derived DNA HBV fragment is inserted in amounts thatare in a high ratio above 100, for example of the order of 1000. Thehigher this ratio, the greater then the number of cells co-transformedby the two vectors at the same time.

It is advantageous to resort to the same basic vectors to form the twotypes of modified vectors used for the co-transformation. A particularlyfavorable basic vector is constituted by the plasmid pBR322. It will bepossible in particular to insert a "DNA HBV tandem" into its EcoRI siteto form one of the modified co-transformation vectors (pCP10).

By the insertion of a tkHSV gene, either into a PvuII site, or into aBamHI site of the same pBR322 plasmid, it is possible to obtain thesecond co-transformation vector (pAG0).

As regards the cultivation conditions, it is naturally self-evident thatthe medium previously used to reach a sufficient development of thecultures, must be replaced at the time of the transformations a littlethereafter by a medium such as the HAT medium in the case where thecomplementation DNA selected is a thymidine-kinase gene, in which thenon-complemented cells are incapable of developing.

The production of the co-transformations, particularly when theabove-said preferred modified plasmids are used in mouse fibroblasts,leads to the remarkable result constituted by the secretion of particleshaving immunological properties characteristic of the envelopes of thehepatitis B virus into the culture medium by the co-transformed cellsnotably these particles are agglutinated by antibodies capable ofagglutinating the natural HBsAg antigens, as isolated from human serum.The presence of secreted particles agglutinatable by anti-HBsAgantibodies can be detected by conventional radio-immunological tests,for example by indirect immunofluorescence using fluorescent anti-HBsAgand anti-IgG rabbit serum.

The amount formed can be measured by direct passive hemagglutinationtests, in manner known in itself for natural HBsAg.

It is also remarkable that the secreting cells, notably in mouse cellcultures, do not contain detectable traces of HBcAg and HBeAg antigens,detectable by direct immunoflurorescence using fluorescent humanantibodies anti-HBc and anti HBe/1, 2, 3. By contacting theco-transformed cells themselves with an anti-HBsAg serum, individualisedcytoplasmic granules are shown by indirect immunofluorescence in a largepart of the co-transformed cells. These particles can also be spottedafter lysis of the cells. In the majority of cases the secretedparticles form the major part of the particles agglutinatable byanti-HBsAg antibodies which can be produced.

The particles formed, whether secreted into the medium or retained inthe cytoplasm of the co-transformed cells, still have the propertieswhich will again be mentioned below in the description of the examples.

It was found however that the transformation of HeLa human cells with acircularised cloned DNA of a hepatitis B virus led to the secretion bythese cells not only of HBs antigen, but also of complete viralparticles containing HBc antigen in the culture medium (9).

Also it is advantageous, according to an additional improvement of theinvention, to resort to an insertion sequence such as has been describedabove, derived from DNA HBV, from which there has previously beenexcised certain portions external to the S gene of the DNA HBV, so as touse only effective genomes which are no longer capable of coding forwhole Dane particles. In particular, recourse is advantageously had toan insertion sequence from which a sufficient portion of the coding genefor the HBc antigen has been deleted, in order to prevent the productionof the latter antigen by the infected cells.

These insertion sequences can consequently be used to modify vectorswhich will then be suitable for transforming eukaryotic cells, whetherit relates to mouse fibroblasts or human cells, but in the absence ofany possibility of HBc antigen production.

In particular, the DNA insertion is advantageously constituted by theDNA of the virus of viral hepatitis B, from which the fragment boundedby the BglII ends at the level of the nucleotides 1986 and 2425 has beendeleted, if reference is made to the diagrammatic map of FIG. 1, insidethe C gene, whose relative position with respect to the whole DNA hasbeen symbolised by the C arrow.

It is also possible to resort to original insertion sequences of DNAHBV, from which larger fragments have been deleted. Notably such asalready defined above.

Of course, this insertion DNA can comprise additional deletions as longas the latter neither affect the expression capacity of the insertionsequence, nor the phases of its translation. In the same way, theinvention extends to the use of any equivalent insertion sequence,whether it relates to the sequence corresponding to a viral DNAbelonging to another sub-type than that which has been contemplated inthe present description and taken up in the examples, or of a sequencefrom which certain parts would have been modified without having alteredthe capacity of the expression products to react immunologically withantibodies against HBs antigens.

It has thus been possible to obtain, from cell cultures of mousefibroblasts transformed with modified vectors by insertion sequences,such as defined above, antigen preparations having the immunologicalcharacteristics of HBsAg antigen, devoid (within the limits of availabledetection methods) of any Dane particle, and of other characteristicantigens contained in the latter, in the absence of components of humanorigin, notably of any serum protein.

In addition, it has been verified that the proteins secreted into theculture media in fact posses vaccinating immunogenic properties, as iswitnessed by their capacity to induce the production of antibodies invivo, that are similar to the human anti-HBsAg antibodies and activeagainst hepatitis virus, more particularly B hepatitis, when they areadministered in vivo, notably to the mouse or the rabbit, according tocurrently used experimental procedures to verify the immunogenicity ofthe natural HBs antigens, extracted from human serums.

The invention hence also relates to novel preparations useful for theconstitution of vaccines, characterised in that they consist of proteinshaving immunogenic and immunological properties characteristic of theHBsAg antigen, having a total purity level as regards the absence of anyDane particle, of HBc antigen and of any serum component of humanorigin.

The invention relates more particularly to vaccine compositionscontaining said antigens (such as can be recovered from culture media ofsaid co-transformed cells or from lysates of the latter), if necessary,associated with any suitable pharmaceutical vehicle for the constitutionof an active vaccine against viral hepatitis capable of beingadministered by the oral or parenteral route.

The invention also relates to laboratory reagents containing particulardoses of these antigens, capable notably of being used as standards orreferences with respect to which the degree of purity of preparationscontaining HBsAg antigens, whether of natural origin or not, can beevaluated as regards in particular their relative content of serumproteins, or other antigens, such as HBcAg or HBeAg, etc.

The invention will again be further illustrated by the description ofexamples of the application of modified plasmids (which themselves formpart of the invention) for the manufacture of antigens havingimmunological properties of HBsAg.

BRIEF DESCRIPTION OF THE DRAWINGS

In this description, reference will again be made to FIG. 1 alreadymentioned and to FIGS. 2a, 2b and 2c, which represent diagrammatic mapsof preferred plasmids which have been utilised according to theinvention.

In the description, the numbers between parentheses refer to thebibliography added at the end of this specification.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE I Construction ofa Recombinant Containing DNA of the Virus of B Viral Hepatitis.

200 ng of pBR322 were subjected to digestion by the endonuclease EcoRIand treated with 2.6 units of alkaline phosphatase in 100 nM ofTris-HCl, pH 8, at 60° C. for 60 minutes. After two extractions withphenol, then three extractions with ether, the DNA was precipitated byethanol. The solid residue was dissolved in water and added to thesolution 100 ng of Eco HBV DNA. The ligation was carried out accordingto the method described in (1).

A culture of E. coli DP 50 followed in a culture medium constituted by abroth L containing 100 μg/ml of diaminopimelic acid and 20 μg/ml ofthymidine.

The bacteria were then transformed according to the above-indicatedmethod by mixing recombinants selected according to their capacity ofwithstanding doses of 100 μg/ml of ampicillin and 15 μg/ml oftetracycline. 900 colonies were obtained. They were all tested byhybridation in situ for the determination of the presence or not of HBVDNA. The test was positive for 800 colonies. The 16 colonies whichinduced a hybridation signal of higher intensity were collected. Theplasmids were extracted and their structure was analysed by digestion inthe presence of EcoRI, XhoI, HindIII and XbaI.

The structure of the recombinant obtained results from FIGS. 2a and 2bin which have been shown diagrammatically, one one hand, the structureof the Eco HBV DNA gene and, on the other hand, the structure of themodified pCP10 plasmid, and such as results from the insertion in theplasmid pBR322 of a DNA fragment constituted by two successive Eco HBVDNA fragments, the head of one being connected to the tail of the other,at the level of an EcoRI site (insertion in tandem head to tail).

It is observed that among the 16 above colonies which were retained, 14among them harbored plasmids of the pCP10 type, in which the two Eco HBVDNA occured inserted according to the head to tail tandem arrangement inthe two directions of orientation possible.

It is in particular due to the digestions with HindIII and XbaIendonucleases that it has been possible to determine the insertiondirections of the Eco HBV DNA fragments Eco.

The integrations in the pCP10 plasmid of two Eco HBV DNA fragments weredemonstrated by digestion in the presence of XhoI endonuclease, whichproduce the excision of an DNA fragment having a size similar to that ofEco HBV DNA from the hybrid plasmid pCP10. The latter includes 10,626pairs of bases.

The respective positions of the S gene in Eco HBV DNA (FIGS. 2a and 2b)and in the hybrid plasmid pCP10 are respectively represented by thearrows S drawn in heavy lines. The small side arrows locate the relativepositions of certain of the restriction sites in the corresponding DNAchains. The positions of the resistance factors to ampicillin (Ap^(R))and to tetracycline (Tc^(R)) are also shown diagrammatically.

The same conventions apply to the pAG0 plasmid (FIG. 2c) which has beenobtained by insertion into the PvuII site of the pBR322 plasmid of thegene of thymidine-kinase of the Herpes simplex virus HSV-1 (HSV-tk) (2).

Cultivation and Transformation by the above-said Plasmids ofThymidine-Kinase Deficient Mouse Cells (Ltk⁺).

Mutants of LM mouse cells deficient in thymidine-kinase (Ltk⁻) werecultivated in the minimum essential medium MEM 0 111 Gibco, if necessaryin the presence of 10% calf serum. Confluent monolayers of these cells(2×10⁶ cells per 24 cm²) in Falcon flasks were inoculated for theirtransformation with DNAs corresponding to the plasmids, according to themethod of GRAHAM and VAN DER EB (3) as modified by STOW and WILKIE (4).

To carry out this transformation there was used at the same time thepAG0 plasmid (linearised by HindIII endonuclease) and pCP10 plasmidlinearised or not by the same enzyme. In all the tests the molecularratio pAG0/pCP 10 has been of the order of 1/1,000. DNA salmon sperm wasused as a vehicle to adjust the concentration of DNA to at least 10μg/ml.

After the transformation, the cell cultures were kept in this mediumcontaining in addition 15 μg/ml hypoxanthine, 0.1 μg/ml of aminopterineand 5 μg/ml of thymidine (selective HAT medium).

24 hours after the transformation, there was added to the medium a HATsolution concentrated 100 times. The concentrated solution was changed aweek later, and then every three days.

After 15 days of cultivation in the presence of the two types ofplasmids (co-transformation) in the selective HAT medium, the formationof colonies was observed. 20 days after the co-transformation, the HBsAgproduction was detected in the culture medium by radio-immunologicaltests. Cultures formed under the same conditions and transformed forpurposes of comparison with the single plasmid pAG0 did not induce anyHBsAg production.

The tk⁺ colonies resistant to the HAT medium were collected with aPasteur pipette (20 days after the co-transformation) and transferred totissue culture on microplates. Passages of the colonies were carried outevery 5 days and they were maintained under continuous selectivepressure in the HAT culture medium.

Five of these colonies obtained from co-transformation in the presenceof linearised pCP10 and 10 colonies obtained from the co-transformationof the mouse cells with the circular pCP10 were sampled and cultivatedin HAT medium. All the cultures produced HBsAg which was released in theexternal culture medium.

The amounts of HBsAg synthesized were variable from one culture to thenext (within limits in a ratio 1 to 30). The amounts produced werestable, even after several passages of said cultures.

The HBsAg can be recovered by centrifugation of the supernatant liquorof the cultures and purified by centrifugation in a density gradientbased on CsCl. The HBsAg were collected in the zone of density 1.20g/ml. These HBsAg were completely neutralised by an anti-HBsAg serumsolution (in a ratio 10/1), after incubation at 37° C. for one hour, asdetected by radioimmunology tests.

On examination under the electron microscope, spherical particles wereobserved having sizes ranging from 18 to 25 nm (on the average 22 nm).Their morphology recalled that of the spherical particles of antigens of22 nm which can be isolated from human serum. Filamentous structuressuch as those visible among the antigens extractable from human serumhave not been observed, at least under the conditions of the experiment.No Dane particle was detected.

The following Table illustrates the production capacity of the mousecells tk⁻ which had been co-transformed by the plasmids concerned.

The ratio P/N corresponds to the ratio of the number of disintegrationsper minute (dpm) measured on the supernatant liquor to the number of dpmmeasured on a controlled culture when the dosages of HBsAg were done byimmunological tests 20 days after the co-transformation of the cells. Aratio P/N higher than 2.1 was considered as significant.

                  TABLE                                                           ______________________________________                                        Co-transformation of Ltk.sup.- cells of mice with                             pCP 10 and pAGO plasmids.                                                                                          number                                                                  salmon                                                                              of                                             linearised                                                                             linearised                                                                             circular                                                                             sperm colonies                                 Flask pAGO     pCP 10   pCP 10 DNA   tk.sup.+                                                                             P/N                               ______________________________________                                        b     0.010    10       --     --    140    24                                c     0.005    --       5      15    200    16                                f     0.010    --       --     15     26     1                                ______________________________________                                    

After lysis of the previously washed cells, digestion of the proteins inthe presence of proteinase, extraction and purification of the cellularDNA, estimation of the cellular DNA by the technique of SOUTHERNmodified by WAHL et al. (5, 6) by using a probe Eco HBV DNA prepared bythe technique of WEINSTOK et al. (7). It has been observed that theamount of intracellular HBsAg which had been synthetisized approximatelycorresponds to a third of the HBsAg which had been excreted into theculture medium.

Under the conditions of the experiment, the most active clones producedup to 150 ng/ml of HBsAg.

In addition to the absence already mentioned of Dane particles, no HBcAgand HBeAg antigens were observed at least at a level detectable bydirect immunofluorescence utilising anti-HBc and anti-HBe/1,2,3fluorescent antibodies of human origin (8). Moreover, the production ofDNA-polymerase, by measurement of the DNA-polymerase activity by themethod of KAPLAN et al., has not been observed (either in thecentrifugation residue from the supernatant liquor or in that of thelysate of the previously transformed cells).

By extraction of the DNAs of high molecular weight of certain clonedproducts, digestion of these DNAs in the presence of EcoRI, Hind III andXhoI, fractionation of the fragments obtained by electrophoresis onagarose gel, transfer to nitrocellulose filters, hybridation of thesefragments with Eco HBV DNA probes labelled with phosphorus 32, it hasbeen established that several copies of the plasmid pCP10 could beincorporated into the cellular DNA. There has also been noted thepresence of HBV DNA dimers both in the plasmid copies contained in thetransformed cells and among the fragments possibly incorporated into thecellular DNAs.

It has also been determined by means of kinetic studies that under theexperimental conditions which have been described, HBsAg antigenparticles can be secreted into the medium by the cells, in theproportion of 2×10⁴ to 4×10⁴ particles/cells/24 hours, namely 2,000,000to 4,000,000 molecules of polypeptide/cells/24 hours, if it is assumedthat each particle contains about 100 molecules of polypeptides.

EXAMPLE II Expression in Eukaryotic Cells of Vectors Modified by anInsertion Sequence Derived from DNA HBV, from which the Major Portion ofthe C Gene has Previously Been Excised.

1) Cloning of pCP9 (Resulting From the Insertion of the LinearisedpBR322 Plasmid in the EcoRI Site of DNA HBV).

Plasmid pBR322 hydrolysed by EcoRI was treated with alkalinephosphatase, then ligated in the presence of an equimolecular amount ofDNA HBV cut by EcoRI. The arrow pCP9 symbolises, in FIG. 1, theinsertion site of pBR322 into the DNA HBV.

2) Construction of Clones of the PAC Series (Containing DNA HBVFragments (Cut by BglII) and PANC (Containing Fragments of DNA HBV cutby BamHI).

The pAG0 plasmid was hydrolysed by BamHI and treated with alkalinephosphatase.

The HBV fragments were obtained from plasmid pCP10 described above,after cleavage by Hind III and Pst I (which have no site in the DNAHBV), and more particularly, from the fragments obtained purified byelectroelution of an agarose gel, and containing a dimer--or a "tandem"sequence--of DNA HBV. These fragments were partially hydrolysed, eitherby the restriction enzyme BglII, or by the BamHI enzyme, under theconditions where only two cleavages were made. The hydrolysates werethen ligated in equimolecular amount with pAG0 plasmid previouslycleaved by BamHI. This takes advantage of the fact that the BamHI andBglII sites are characterised by common cohesive end portions.

A collection of clones sensitive to tetracycline was obtained andanalysed in order to isolate each insertion possibility.

Clones PAC 12, PAC 14, PAC 16 and PANC 34 were obtained. The arrowsaccompanying the corresponding symbols in FIG. 1, locate the insertionsof the linearised pBR322 in the BamHI or BglII sites corresponding tothe sequences of DNA HBV contained in the recombinant plasmids obtained.

These clones had in addition the following characteristics:

PAC 12: deletion of the BglII fragment 1985-2840;

PAC 14: deletion of the BglII fragment 1986-2425;

PAC 16: insertion of the genome of pBR322 in the 1986 site of the DNAHBV;

PAC 34: it contained the pBR322 genome in the BamHI 1400 site of DNAHBV.

These clones have been used to transform LTK⁻ cells in the proportionsof 2 μg of plasmid per 2.10⁶ cells. 4 weeks later, the TK⁺ coloniesreached confluence. The culture medium was then analysed for thepresence of HBs by applying the above-said radio-immunological tests.

The capacities or non-capacities of the various recombinant plasmids toexpress the S gene, in the form of HBs antigen secreted into the culturemedium, have been expressed respectively in FIG. 1 by the signs "+" and"-".

The expression of the S gene in the clones PAC 14 and PAC 16 and thefailure of expression in the clone PAC 12 show that the transcription ofthe S gene is initiated in viral DNA and more precisely in therestriction fragment BglII 2425-2840. There exists a sequence TATATAAalso called "TATATAA Box" situated at 72 nucleotides upstream of thebeginning of the "pre-S" region which appears to control thetranscription of the S gene.

The fact that no expression was obtained with the PANC 34 clone, underthe conditions in which the experiment was carried out, is perhapsconnected with the fact that the DNA encoding for the mRNA stops afterthe BamHI site.

It is particularly most significant that the PAC 14 clone, whichproduces the HBs, possesses a deletion in the C gene of the virus, whicheliminates the risk of the production of viral particles for thetransformed cell, whatever its nature, and permits its use for theproduction of a vaccine.

The invention hence provides a product capable of a purity unattainablehitherto. It relates more particularly to the preparations of proteinparticles having the immunological properties of HBsAg, essentially freeof serum protein. They are totally free of Dane particles detectable bythe usual methods of radio-immunological measurement. They are inaddition totally free of proteins, notably serum proteins of humanorigin. They are free of DNA polymerase.

The invention relates also again to the novel DNA insertion sequencesthemselves, as they have been defined above, and the vectors, notablyplasmids, containing said sequences which are more particularlycharacterised by the fact that they contain the promoter of thetranscription of the S gene. These vectors are of very particularinterest in that they can if necessary be modified by the insertion of aparticular DNA sequence corresponding to a protein whose expression intoeukaryotic cells is sought. In fact, this vector has a particularinterest in not being toxic with respect to the cells concerned. Thepromoter is not repressed, since these cells permit the expression ofthe S gene. In addition, this vector can enable the secretion ofproteins synthesized directly in the culture medium.

One of the above-said novel insertion sequences can again be defined ascontaining, in addition to the S gene, the DNA of the "pre-S" region(which in the DNA of the virus of viral hepatitis is situatedimmediately upstream of the S gene in the reverse direction of thetranscription, this gene including of the order of 163 nucleotides), thegene of the messenger RNA of the S gene of the virus of B hepatitis, thepromoter of the DNA of the virus of B hepatitis, notably a TATATAAsequence (T being abbreviation for thymine and A abbreviation foradenine). It comprises notably also a DNA sequence corresponding to asequence localised between the nucleotide positions 2425 and 2840 of theDNA of the virus of B hepatitis.

There are obviously included within the scope of the invention,sequences including several units of the above-described type, notably"tandem" sequences.

The capacity of the vectors modified according to the invention of beingexpressed in eukaryotic cells seems to witness of the fact that thetranscription of the gene of HBsAg is under the control of a viralpromoter contained in the fragment derived from HBV DNA. These vectors,such as the plasmid pCP 10, PAC 14 or PAC 16, can themselves be used asvectors for effecting the expression in eukaryotic cells of a foreignDNA previously inserted into said vectors. It appears that the advantageof the vectors modified according to the invention must reside in thefact that they do not induce lysis of the host-cells. Moreover the Sgene can be considered as having a sequence signal under the control ofwhich the hybrid protein formed, resulting from the expression into thehost cells of foreign DNA previously inserted into the above-saidvector, would be excreted into the culture medium. The recovery of thishybrid protein would obviously be considerably facilitated therefrom.

Although the method of the invention has been described in theforegoing, principally in its application to DNA HBV, it can be extendedto any other circular DNA or whole genomes. In particular it can beapplied to the study of circular DNAs of "non A", "non B" viruses of thecorresponding viral hepatitides.

Below the bibliography relating to the state of the art and to whichreference is made in the description of the examples, is given:

(1) TIOLLAIS, P., PERRICAUDET, M., PETTERSON, U. & PHILIPSON, L. (1976),Gene 1, 49-63.

(2) COLBERE-GARAPIN, F., CHOUSTERMAN, S., HORODNICEANU, F., KOURILSKY,P. & GARAPIN, A. C. (1979), Proc. Natl. Acad. Sci. U.S.A., 76,3755-3759.

(3) GRAHAM, F. L. & VAN DER EB, A. J. (1973), Virology 52, 456-458.

(4) STOW, N. D. & WILKIE, N. M. (1976), J. Gen. Virol. 33, 447-458.

(5) SOUTHERN, E. M. (1975), J. Mol. Biol. 98, 503-517.

(6) WAHL, G. M., STERN, M. & STARK, G. R. (1979), Proc. Natl. Acad. Sci.U.S.A. 76, 3683-3687.

(7) WEINSTOCK, R., SWEET, R., WEISS, M., CEDAR, H. & AXEL, R. (1978),Proc. Natl. Acad. Sci. U.S.A. 75, 1299-1303.

(8) TREPO, C., HANTZ, L., VITVITSKI, L., CHEVALLIER, P., WILLIAMS, A.,LEMAIRE, J. M. & SEPTJIAN, M. (1978) in Viral Hepatitis, eds. VYAS, G.N., COHEN, S. N. & SCHMID, R. (The Franklin Institute Press) pp.203-209.

(9) SHALOMZ. HIRSCHMAN and Coll., Proc. Natl. Acad. Sci. U.S.A., vol.77, N°9, pp. 5507-5511, September 1980, "Expression of cloned hepatitisB virus DNA in human cell cultures".

We claim:
 1. A recombinant DNA molecule, wherein said recombinant DNAmolecule comprises:more than two Hepatitis B virus (HBV) DNA sequencesrecombined with a vector, wherein said vector comprises sequences of E.coli plasmid pBR322 and has an antibiotic resistance gene as a selectivemarker, wherein each of said HBV DNA sequences is comprised offull-length HBV DNA, and wherein said HBV DNA sequences are arranged ina head-to-tail tandem relationship.